A Smartphone-Enabled Colorimetric Platform Based on Enzyme Cascade Amplification Strategy for Detection of Staphylococcus aureus in Milk

Staphylococcus aureus (S. aureus) is a pathogenic bacterium-contaminating milk and dairy foods causing food poisoning and foodborne pathogens. In this work, a smartphone-enabled enzyme cascade-triggered colorimetric platform was constructed using cascade bio-nanozyme formed by immobilized glucose oxidase (GOx) on the Fe 3 O 4 @Ag for rapid detection of S. aureus. Benefiting from reasonable experimental design, a bio-nanozyme cascade-triggered reaction was achieved through H 2 O 2 produced by GOx oxidation of glucose, followed by in situ catalysis of 3,3 ′ ,5,5 ′ -tetramethylben- zidine (TMB) by the inherent peroxidase-like activity of Fe 3 O 4 @Ag to produce color signals. S. aureus detection could be performed through naked-eye observation and smartphone measurement, the developed assay can achieve quantitative and qualitative detection of S. aureus. The on-site nanoplatform had satisfactory specificity and sensitivity with a low detection limit of 6.9 cfu·mL −1 in 50 min. Moreover, the nanoplatform has good practicality in the detection of S. aureus in milk samples. Therefore, the assay has potential application prospects in food safety inspection.


INTRODUCTION
Staphylococcus aureus (S. aureus) is a Gram-positive, hazardous, and widely distributed foodborne pathogen (Argudín et al., 2012), which has posed a serious threat to human health and global food safety (Ramessar and Olaniran, 2019).Especially in developing countries, due to the consumption of raw milk and poor hygiene in milk and dairy production environments, S. aureus can cause ptomaine poisoning, diarrhea, and staphylococcal septicemia (Wang et al., 2022b).Millions of people worldwide are infected with foodborne S. aureus every year, and its fatality rate is 0.03% (Hennekinne et al., 2012).This indicated the need to develop a straightforward, highly responsive, and precise technique for onsite detection of S. aureus in the food control process.
Currently, detection methods of S. aureus and other foodborne pathogens usually depend on bacteria culturing (Chen et al., 2022), which is cumbersome and time-consuming.It usually takes 3-5 d to get detection results (Rubab et al., 2018, Samani et al., 2021).Due to technological advancements, a range of state-of-the-art methods have been employed for the detection of S. aureus., such as enzyme-linked immunosorbent assay (ELISA) (Bourhy et al., 2013) and polymerase chain reaction (PCR) as replacements for culturing (Yuan et al., 2018).Although these technologies have significantly enhanced sensitivity and specificity, they continue to encounter challenges: poor cost-effectiveness and are not suitable for average users.Recently, research has shifted toward the development of rapid and on-site assays, such as paper-based methods (Kim and Oh, 2019), Lateral flow assays (LFAs) (Mahmoudi et al., 2020) and Microfluidic paper-based analytical devices (Pang et al., 2018), however, these detection limits are often still either above the infectious dose or the assay has become increasingly complex, which limits on-site use.Thus, it is critical to develop a new rapid and sensitive strategy in food analysis.
In comparison to other approaches, a colorimetric assay based on nanomaterials and enzyme catalytic performance is one of the most sensitive approaches, which can obtain test results with the naked eye and smartphones.This method not only cuts down on time and costs but also facilitates real-time analysis and on-site testing as needed.(Zarei, 2017, Zhang et al., 2018a).Magnetic nanomaterials, such as Fe 3 O 4 nanoparticles (Fe 3 O 4 NPs), are widely used in biological and medical colorimetric detection fields due to easy modification and bio-nanozyme activity (Wu et al., 2018).Fe 3 O 4 NPs and related Fe 3 O 4 composite nanomaterials (such as Fe 3 O 4 @Ag NPs) can catalyze substrate H 2 O 2 and further oxidize TMB to generate color signals, to achieve colorimetric detection.However, H 2 O 2 is easily decomposed and has poor stability, which is not suitable for on-site detection methods.To address this key point, the enzyme cascade reaction, as a highperformance amplification and visualized methodology, was employed to avoid the instability of intermediates or substrates (Zhao et al., 2018).By cascade reaction, enzymes are densely organized within the same scaffold, creating a multi-enzyme complex that allows efficient communication between enzymes, as it channels the product of one enzyme to another as a substrate.This arrangement enhances catalytic efficiency when compared with individual enzymes working in isolation.(Zhang et al., 2018b, Zhao et al., 2018).The detection signal can be amplified several orders of magnitude in a short time by enzyme cascade reaction, which is beneficial for shortening testing time and developing on-site testing methods (Guo et al., 2020).
Due to nanozymes having good chemical stability similar to natural enzymes and better anti-interference, immobilized natural enzymes with nanozymes by covalent binding or embedding to construct bio-nanozymes will be a breakthrough in improving enzyme stability and constructing new on-site detection method (Cao et al., 2022, Zhang et al., 2022).Among Fe 3 O 4 composite nanomaterials, Fe 3 O 4 @Ag NPs play an important role in bio-nanozymes, their inherent magnetic properties can quickly capture and isolate target bacteria with the modification of aptamer by external magnetic fields, and the Ag shell of nanoprobes have a large specific surface area, good catalytic activity, and easy functionalization properties (Gao et al., 2015, Meng et al., 2015).According to previous reports, the Ag shell of Fe 3 O 4 @Ag nanocomposites can improve the activity and stability of the natural enzymes in different pH and temperatures (Hashemifard et al., 2010), which is a promising bio-nanozyme for further establishing a novel on-site method for S. aureus detection.
Herein, we have developed a user-friendly and integrated multifunctional nanoplatform for the detection of S. aureus detection based on an enzyme cascadetriggered reaction coupled with a smartphone.Using Fe 3 O 4 @Ag NPs as a scaffold, Glucose oxidase (GOx) and the aptamer of S. aureus were immobilized on its surface by a simple freeze-thaw method to synthesize Fe 3 O 4 @ Ag -GOx/ aptamer.Compared with traditional bio/nano interface construction methods, this method is a simple and efficient labeling method without reagents.This occurs because growing ice crystals exclude nanoparticles, thereby screening the electrostatic repulsion between them.Consequently, the local concentration of the polymer around the nanoparticles also increases.(Liu and Liu, 2017).The process of the efficient enzyme cascade system for detecting S. aureus is shown in Scheme 1.The diffused glucose was catalyzed by GOx to produce H 2 O 2 , which was further oxidized TMB by the peroxidase-like catalytic activity of Fe 3 O 4 @Ag, thereby achieving the color changes of TMB solution.In the presence of S. aureus, Fe 3 O 4 @ Ag nanoprobes can capture S. aureus specifically to prevent glucose from binding to the catalytic active site of GOx.The inhibition of the enzyme cascade catalytic activity is related to the color change of the substrate (TMB), and the intensity of the color was closely related to the number of S. aureus.Meanwhile, according to the RGB values, using the smartphone APP to analyze the obtained images can timely obtain the danger level of the samples, which can achieve on-site detection.

EXPERIMENTAL SECTION
Materials and reagents, apparatus, bacteria culture, the preparation of Fe 3 O 4 @ Ag -GOx/ aptamer nanoprobes, and detailed steps for steady-state kinetics and optimizing experimental conditions can be found in Supporting Information (SI).

PROOF-OF-CONCEPT SMARTPHONE APPLICATION DEVELOPMENT AND VERIFICATION
We developed a prototype smartphone application (APP) using the Android Studio system, implementing a Java algorithm for extracting images and analyzing them.The code of the APP is described in the SI.The APP operates through the following steps: (1) Click the "analyze" button within the application; (2) Choose the captured image of the results; (3) Select the detection region (center of the test tube).The RGB color model is employed to analyze the color of the selected area.APP will produce Safe, Danger, and Extreme Danger

ACTUAL SAMPLES TESTING
To validate the applicability of our developed method in real sample detection, we utilized pure milk purchased from a local supermarket in Changchun, China, as the food matrix.These samples were used to create the research model for detecting S. aureus in actual food samples.Specifically, the plate-culture method was employed to verify whether the 2 samples contained bacteria, and then 500 μL of the cultured S. aureus solution (10 2 , 10 4 , and 10 6 cfu/mL) was added to 6 mL milk samples to construct food samples containing bacteria, respectively.We also subjected the same samples to the plate counting method for comparison, ensuring the reliability of our developed detection method.

Characterization of Fe 3 O 4 @ Ag -GOx/ aptamer
The structure and morphology of the synthesized Fe 3 O 4 @Ag NPs and Fe 3 O 4 @ Ag -GOx/ aptamer were investigated by Transmission Electron Microscope (TEM), Vibrating Sample Magnetometer (VSM), Fourier Transform Infrared Spectroscopy (FTIR), x-ray Diffraction (XRD), and Energy Dispersive x-ray Spectroscopy (EDS) Mapping.As shown in Figure 1a, the original Fe 3 O 4 @Ag NPs (Fig. S1) and Fe 3 O 4 @ Ag -GOx/ aptamer NPs exhibited a uniform and typical spherical with a smooth surface (Figure 1a), while the hydrated particle size increased from 268.3 ± 23.1 nm to 830.7 ± 121.6 nm, respectively (Fig. S2).After modifying the GOx and aptamer, the UV spectrum exhibits a significant red shift (Fig. S3).The Zeta potential of Fe 3 O 4 @Ag NPs and Fe 3 O 4 @ Ag -GOx/ aptamer NPs was −29.50 mV and −32.60 mV (Fig. S4), respectively.The saturation magnetization values of Fe 3 O 4 @Ag NPs and Fe 3 O 4 @ Ag -GOx/ aptamer NPs have nearly zero coercivity at room temperature (Figure 1b), which enables rapid and efficient separation under the influence of an external magnetic field.The FTIR spectrum indicates obvious characteristic bands at 585 cm −1 , 1633 cm −1 , and 3432 cm −1 (Figure 1c), it demonstrated the stretching vibration of the Fe-O band, O-H/N-H group, and C = O group and successful modification of aptamer and GOx (Wu et al., 2015, Lou et al., 2021).XRD results depicted in Figure 1d above results indicate that synthesized nanomaterials containing Fe, Ag, O, C, and N elements have been successfully prepared.In addition, Fig. S5 confirmed that Fe 3 O 4 @ Ag -GOx/ aptamer can capture S. aureus to form Fe 3 O 4 @ Ag -GOx/ aptamer -S.aureus complex, which can be used to construct enzyme cascade-triggered colorimetric assay.

Feasibility verification
To verify the feasibility of the detection system, the peroxidase-like catalytic activity of Fe 3 O 4 @Ag, Fe 3 O 4 @ Ag -GOx, Fe 3 O 4 @ Ag -GOx/ aptamer and Fe 3 O 4 @ Ag -GOx/ aptamer + S. aureus was first evaluated in the reaction of TMB with glucose.As shown in Figure 2, the naked Fe 3 O 4 @Ag NPs without modified GOx cannot hydrolyze glucose to produce H 2 O 2 , therefore, unable to oxidize the substrate TMB, and the UV absorption peak at 450 nm is negligible.After the modification of GOx, TMB can be catalyzed to produce an obvious color change and a raised UV absorption peak can be observed, which indicates that the GOx-Fe 3 O 4 @ Ag enzyme cascade reaction system was constructed successfully.According to the orange line and blue line, modification of the aptamer on Fe 3 O 4 @ Ag -GOx did not affect the enzyme cascade activity of the prepared nanomaterials, which can be used to construct a detection system.In the presence of S. aureus (pink line), the UV absorption peak at 450 nm was significantly reduced, due to the blocking of catalytic sites caused by the binding of target bacteria.According to the above results, the successfully constructed enzyme cascade reaction system can be used as a colorimetric detection sensor for S. aureus.

Steady-state kinetic of Fe 3 O 4 @ Ag -GOx/ aptamer
The catalytic activity of Fe 3 O 4 @Ag, Fe 3 O 4 @ Ag -GOx, Fe 3 O 4 @ Ag -GOx/ aptamer, and Fe 3 O 4 @ Ag -GOx/ aptamer + S. aureus was further investigated using TMB and H 2 O 2 as substrates by measuring the absorbance change at 652 nm in time-drive mode (Figure 3).The Michaelis constant (K m ) was determined by plotting the double reciprocal of the Michaelis-Menten equation using Lineweaver-Burk plots.: where V is the initial velocity, V max is the maximum velocity, and [S] is the substrate concentration, respectively.According to Table S1, the K m value of Fe 3 O 4 @ Ag, Fe 3 O 4 @ Ag -GOx, and Fe 3 O 4 @ Ag -GOx/ aptamer all similar and all lower than Fe 3 O 4 @ Ag -GOx/ aptamer + S. aureus.This indicates that the affinity of Fe 3 O 4 @ Ag -GOx/ aptamer + S. aureus composite for TMB and H 2 O 2 is significantly decreased because of the binding of target bacteria and nanomaterials.In addition, the V max also decreased significantly after the bacteria were captured by the Fe 3 O 4 @ Ag -GOx/ aptamer probe.These results further prove that the target-induced shielding effects are caused by the inhibition of catalytic activity of Fe 3 O 4 @Ag by S. aureus.
In addition, the stability of Fe 3 O 4 @ Ag -GOx/ aptamer at different temperatures and pH in the TMB-H 2 O 2 system and the TMB-glucose system was also investigated.According to Fig. S6, the catalytic activity of Fe 3 O 4 @ Ag -GOx/ aptamer NPs has good stability at low temperatures (4°C -37°C) in the TMB-H 2 O 2 system, while their absorbance values significantly decreased above 60°C, due to the decomposition of H 2 O 2 caused by high temperature.Under acetic conditions (pH = 2.0 -6.0), the catalytic activity of Fe 3 O 4 @ Ag -GOx/ aptamer NPs was stable, due to the instability of H 2 O 2 under alkaline conditions, the absorbance values were decreased when the pH values exceeded 8.0.(Zhang et al., 2009).In the TMB-glucose system (Fig. S7), high temperature and extreme pH values can cause the decomposition of H 2 O 2 and GOx inactivation.Therefore, 25°C and pH = 4 were the optimal conditions for Fe 3 O 4 @ Ag -GOx/ aptamer and the enzyme cascade reaction system of GOx-Fe 3 O 4 @Ag to maximize catalytic activity.

Optimization of experimental conditions
The experimental conditions of this method were fine-tuned to achieve optimal detection performance.All experimental details and results are illustrated in

Analytical performance
Under optimized conditions, the absorbance of this assay was measured by a series concentration of S. aureus (10-10 7 cfu•mL −1 ) for sensitivity analysis.According to Figure 4a and b, as the concentration of S. aureus increased (from 10 to 10 7 cfu•mL −1 ), both the color intensity and the related absorbance at 450 nm gradually decreased in the sensing system.The normalized A 450nm for various concentrations of S. aureus was calculated as follows (Yao et al., 2020):  where A 450nm is the absorbance of various S. aureus concentrations, and A max and A min are the maximum and minimum values at A 450nm , respectively.The A 450nm exhibited a good linear relationship with the log scale of S. aureus concentration in the range of 10-10 7 cfu•mL −1 (Figure 4c), and the linear regression equation is: y = −0.135logC+ 0.994, with R 2 is 0.984, and the limit of detection (LOD) was 6.9 cfu•mL −1 (based on the 3S/slope, where S is the standard deviation of blank control).In addition, intra-day and inter-day stability of the system was evaluated at 3 different concentrations (1 × 10 2 , 1 × 10 4 and 1 × 10 6 cfu•mL −1 ).The relative standard deviation (RSD) for detecting S. aureus ranged from 0.9% to 5.96% (Table S3), indicating that the S. aureus detection method based on enzyme cascade reaction had good intra-day and inter-day stability.
Using this procedure, the target bacteria were adjusted to a concentration of 10 5 cfu•mL −1 , and 4 nontarget bacteria, L. monocytogenes, E. coli O157:H7, V. parahaemolyticus, and S. typhimurium were set at 10 6 cfu•mL −1 , to validate selectivity of this assay.As shown in Figure 4d, we could detect the presence of target bacteria, and the presence of non-target bacteria did not affect the accuracy of this assay.These results indicated that the Fe 3 O 4 @ Ag -GOx/ aptamer nanoprobe could only specifically capture S. aureus.
In addition, we further developed a fast and convenient smartphone platform to satisfy the requirement of on-site analysis.The gray value of detection system images can be analyzed by using the established proof-of-concept image analysis APP.After calibration (Table S2), as the concentration of S. aureus increased, the color of the detection system gradually became lighter, and the gray value obtained by this method also gradually decreased.Therefore, the gray value can be translated into a semiquantitative analysis of S. aureus.The analysis standard is shown in Table S3, and our APP can directly determine the danger level of the obtained image results (Figure 5).
Based on the above results, our enzyme cascade-triggered colorimetric assay has good sensitivity, selectivity and precision (Table S4) for S. aureus detection and after integration with smartphone APP, it can realize visual detection within 50 min and real-time analysis without relying on the instrument.

Analysis of real samples
The practicability of the proposed assay was evaluated by using artificially contaminated milk samples.Food samples are analyzed and compared with the plate-counting method (Fig. S9) and real-time PCR assay (Fig. S10).The LOD of qPCR for S. aureus detection was 10 4 cfu•mL −1 , and the detection time of the entire method is 120 min (52 min for DNA extraction, and 68 min for PCR amplification).According to Table S5, all the average recoveries for various concentrations in food samples were in the range of 98.01% -101.33%, and the RSD ranged from 4.44% to 7.77%, indicating that our method has strong anti-interference ability.

DISCUSSION
In this work, a smartphone-enabled enzyme cascadetriggered colorimetric platform was constructed using cascade bio-nanozyme for rapid detection of S. aureus.Nanozymes are ideal for recognition units due to their stability, cost efficiency, and easy synthesis.Recent studies have demonstrated the successful application of nanozyme-based assays for detecting a wide range of targets, including toxic ions, phenolic compounds, pesticide residues, antibiotic residues, and pathogenic bacteria.The development of smartphone-based sensors that are miniaturized, integrated, and provide rapid feedback is attracting a lot of interest, especially in the field of food safety detection.Changes in color, fluorescence, or other properties of nanozyme-based systems can indicate analyte levels, allowing for con- venient quantitative analysis through a smartphone APP (Sun et al., 2021, Liu et al., 2023).For instance, a kind of colorimetric aptasensor has been established for detecting enrofloxacin (Zhu et al., 2022a) and phenolic compounds (Zhu et al., 2022b) based on single peroxidase-mimic nanozyme using a smartphone, which could be achieved because the nanozyme activity was different in the presence and absence of the target.However, the amplification efficiency of a single enzyme reaction is limited.Enzyme cascade which embraces multiple enzymatic reactions can achieve extremely high specificity and catalytic efficiency because they can limit the accumulation of undesired intermediaries and prevent unproductive cross-talk (Xu et al., 2024).Due to these advantages, an enzyme cascade reaction by bio-nanozyme was designed for developing sensitive and selective bioanalysis methods in this work.A simple freeze-thaw method was used to immobilize glucose oxidase (GOx) and the aptamer of S. aureus on Fe 3 O 4 @Ag NPs to form the bio-nanozyme.Diffusion of glucose was catalyzed by GOx to produce H 2 O 2 , which was further oxidized by Fe 3 O 4 @Ag on TMB to produce the color changes.Currently, the most frequently used analytical tools for signal processing have been the fluorescence sensor (Bagheri Pebdeni et al., 2021, Li et al., 2023), colorimetric sensor (Wang et al., 2022a), RPA-CRISPR (Ma et al., 2024), and immunofluorescence sensor (Cui et al., 2021).The sensitivity of the above studies for detecting S. aureus is compared in Table 1.The different detection methods had diverse detection time from 40 to 120 min, so the detection time of 50 min in our research is acceptable.And the LOD of enumerated method was 10 -10 3 cfu•mL −1 , our assay has the lower LOD (6.9 cfu•mL −1 ).These analytical tools had a similar detection range for S. aureus in practical samples, but our smartphone-based assay is portable and integrated, which is more suitable for on-site detection.
Additionally, a smartphone-enabled colorimetric platform offers several practical advantages.(1) Our materials are readily available.Different from the preparation of materials that require chemical or functional group modification, it can be obtained Fe3O4@ Ag materials using a simple solvothermal method without any additional materials being introduced.(2) Our method offers a higher sensitivity, which is attributed to the lower Michaelis constant of bio-nanozyme.After combining with GOx, our bio-nanozyme has better catalytic activity, which is an efficient signaling method by cascade enzyme reaction and ensured the sensitivity Image and 4b UV-Vis spectrum of sensing system with various concentrations (0, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 cfu•mL −1 ) of S. aureus.4c the calibration plot for S. aureus (normalized A 450nm vs. the log scale of S. aureus concentration).4d Images and normalized A 450nm of the presented colorimetric method corresponding to different bacteria (1 → 7: blank, S. typhimurium, V. parahaemolyticus, L. monocytogenes, E. coli O157:H7, S. aureus and S. aureus + mixture, mixture consists of a mixture of S. typhimurium, V. parahaemolyticus, L. monocytogenes, and E. coli O157:H7).
of the detection method.(3) The smartphone APP can enhance the portability of the detection assay.Hence, the smartphone-enabled colorimetric platform is easily usable in resource-limited areas.Additionally, it has broad prospects for the detection of foodborne pathogens across a wide range of species by simply changing the aptamer, which is expected to be to monitor global food safety.

CONCLUSIONS
A new enzyme cascade-triggered strategy for colorimetric assay of S. aureus with high sensitivity was constructed.The sensing system delivers good sensitivity with a LOD of 6.9 cfu•mL −1 and was integrated with a smartphone platform we designed to achieve directly determining targets.The smartphone platform is ideal for on-site analysis, eliminating the need for additional instruments, moreover, even in complex food matrices, it can convert and amplify signals in one step for sensitive and selective detection of foodborne pathogens.Future research could focus on developing methods for detecting multiple types of foodborne pathogens, thereby enhancing food safety and hygiene standards.
Wang et al.: A Smartphone-Enabled Colorimetric… based on the sample's safety level, allowing for realtime semiquantitative analysis.
Scheme 1. Schematic illustration of S. aureus detection based on enzyme cascade-triggered colorimetric reaction.

Figure 5 .
Figure 5. Semiquantitative analysis of S. aureus using smartphone APP.